Various kinds of methods, substances and approaches have been conventionally known for extraction or purification of a particular objective protein from other components in a sample, such as bacteria, yeast, insect cells, animal cells, animal tissues, plant tissues (including lysate, extract and the like obtained from these), cell-free protein synthesis solutions and the like (hereinafter these are generally referred to as a “biological sample”).
One example of general approach utilizes nonspecific affinity of a protein for a carrier. As such approach, for example, ion exchange chromatography based on the electric charge of protein molecule is known. In ion exchange chromatography, a protein mixture is added to a chromatography matrix having an opposite electric charge from the protein, and various proteins are allowed to bind with the matrix by reversible electrostatic interaction. The protein bound with the matrix can be eluted in the order of from one having a weaker bond to one having a stronger bond, by increasing the ionic strength or changing the pH of the elution buffer.
An example of other general approach is one utilizing the physical property of the protein as a means for separation. This approach is represented by known gel filtration based on the size of protein. In gel filtration, a protein mixture is applied to a gel filtration column packed with a matrix for chromatography having a given size of pores. Thereafter, elution is done using an eluent (generally buffer) to obtain individual chromatography fractions, which are subjected to analysis.
A still another example of the general approach is one utilizing specific affinity of protein for a reagent for purification. Known examples of such approach include affinity chromatography utilizing an antibody capable of specific adsorption to the objective protein, and, when the objective protein is an antibody, an antigen capable of specific adsorption to said antibody. In the affinity chromatography, generally, antibody or antigen is bound with a column substrate, and a solution containing an antigen or antibody capable of specifically adsorbing to said antibody or antigen is applied to the column, thereby to form an immune complex (antigen-antibody complex) on the column substrate. Subsequently, for example, the above-mentioned immune complex is exposed to a buffer having an extremely high ionic strength or a buffer having an extremely high or low pH buffer to instabilize the immune complex into elution. Thus, affinity chromatography is a highly effective purification method of protein based on the specific interaction between the objective protein and ligand immobilized on the solid phase. In affinity chromatography, generally, a solid phase ligand has several inherent scientific properties and due to such properties, it affords selective adsorption of the objective protein. Contaminant proteins can be removed because they do not bind with a solid phase ligand or by washing the solid phase ligand with a suitable solution.
The possibility of preparation of hybrid gene by recent gene technology has opened a new avenue. That is, by binding a gene sequence encoding a desired protein with a gene sequence encoding a protein fragment (affinity peptide) having high affinity for the ligand, a recombinant protein having an affinity peptide suitable for the above-mentioned separation can be expressed, which in turn has made it possible to purify a desired recombinant protein in the form of a fusion protein by a single step of purification using the affinity peptide. In addition, by mutation restricted to a certain site, a specific chemical or enzymatic cleavage site can be introduced into the binding site of the affinity peptide and the desired recombinant protein, in which case the fusion protein is purified using a suitable affinity resin and the affinity peptide is cleaved chemically or enzymatically to recover the desired recombinant protein. Such purification method is known from, for example, Science 198, 1056-1063 (1977) (by Itakura et. al), Proc. Natl. Acad. Sci. U.S.A. 80, 6848-6852 (1983) (by Germino et al.), Nucleic Acids Res. 13, 1151-1162 (1985)(by Nilsson et. al.), Gene 32, 321-327 (1984) (by Smith et al), U.S. Pat. No. 5,284,933 and U.S. Pat. No. 5,643,758.
The affinity peptide of fusion protein can be directly or indirectly bound with a biochemically active polypeptide or protein. When a single affinity peptide is used, the affinity peptide can be bound with the amino terminal amino acid or carboxyl terminal amino acid of a biochemically active polypeptide or protein. When two affinity peptides are used, one of the affinity peptides may be bound with the amino terminal amino acid of a biochemically active polypeptide or protein and the other affinity peptide may be bound with the carboxyl terminal amino acid of the biochemically active polypeptide or protein.
In the case of an indirect binding, the affinity peptide contains a suitable selective cleavage site and can be bound with a desired biochemically active polypeptide or protein via said selective cleavage site. Known preferable selective cleavage sites are amino acid sequences -(Asp)n-Lys- wherein n is 2, 3 or 4 and -Ile-Glu-Gly-Arg-. These selective cleavage sites can be specifically recognized by enterokinase and aggregating factor Xa, respectively, which are proteases. Such affinity peptide can be enzymatically cleaved at the above-mentioned selective cleavage sites by a method known per se.
In the case of direct binding, the affinity peptide remains bound with a desired biochemically active polypeptide or protein. That is, the affinity peptide does not have a chemically or enzymatically cleavable selective cleavage site. The direct bond is advantageous when the activity of the desired polypeptide or protein is not adversely affected by the presence of affinity peptide.
U.S. Pat. No. 5,643,758 discloses a binding protein having an affinity peptide having specific affinity for particular carbohydrate; in other words, a production method of a fusion protein using a carbohydrate-binding protein and a convenient purification method using it. The above-mentioned carbohydrate-binding protein is exemplified by mono, di or polysaccharide-binding protein, which is more specifically a maltose-binding protein, an arabinose-binding protein or the like. Particularly, the maltose-binding protein, which is a malE gene product of Escherichia coli, is a periplasm protein subject to an influence of osmotic pressure, and shows specific affinity for maltose and maltodextrin. This maltose-binding protein, its fragment, and a fusion protein with an objective protein can be purified by affinity column chromatography using an amylose resin. Those of ordinary skill in the art know that utilization of a maltose-binding protein as an affinity peptide of fusion protein leads to a high possibility of purification in a solubilized state, even when the objective protein is sparingly soluble or easily insolubilized.
As mentioned above, there are various purification methods of protein, among which affinity column chromatography utilizing fusion protein is particularly a highly superior purification method. These methods generally include regeneration and recycled use of expensive materials used, such as carrier and the like, which renders it not entirely convenient. The conventional purification method mainly based on column chromatography requires a lot of time for sequentially passing samples through column, due to which automation and high throughput are difficult to achieve.
The present invention has been made to solve the above-mentioned problems and provides a purification method of protein, which is strikingly convenient as compared to conventional methods and which permits automation and high throughput, and materials therefor.